Project Summary/Abstract Acute kidney injury (AKI) confers high morbidity and mortality. Even though patients survive, there is a substantial risk of progression to chronic kidney disease (CKD). Unfortunately, there is no effective therapy to retard this progression. ?Klotho (here referred as Klotho) was shown to effectively block this transition. But, whether Klotho?s renoprotection involves prevention of vascular rarefaction and whether the suppressive effect of Klotho on renal fibrosis is associated with the clearance of collagen are largely unknown. Abnormal autophagy may lead to renal tubular and vascular damage, and renal fibrosis. Vascular rarefaction and renal fibrosis contribute to CKD. Klotho deficiency is associated with impaired angiogenesis. But, it is unclear whether Klotho can rescue peritubular capillaries in AKI. Klotho was found to upregulate autophagy, improve revascularization, and promote collagen I degradation in vitro; but, how Klotho affects autophagy and if this effect modulates vascular rarefaction and fibrosis, and prevents progression to CKD needs to be defined. The central hypothesis is that Klotho prevents AKI transition to CKD by promoting autophagy, which in turn inhibits renal fibrosis and ameliorates vascular rarefaction. There are three specific aims to test this hypothesis. Aim 1 investigates if Klotho increases autophagy flux in the kidney by (1) examining if Klotho upregulates renal autophagy flux in LC3 reporter mice; (2) defining the mechanisms of upregulation of autophagy by Klotho in vivo; (3) exploring how Klotho regulates autophagy flux in kidney cell line. Aim 2 examines if Klotho inhibits renal fibrosis via upregulation of autophagy activity by (1) examining if Klotho upregulates autophagy and inhibits renal fibrosis post-IRI; (2) defining if autophagy levels influence renal fibrosis post-AKI using mice with low or high autophagy activity; (3) investigating how autophagy reduces collagen I or promotes the degradation of collagen I in GFP-collagen I transfected cells in vitro. Aim 3 explores if Klotho ameliorates vascular rarefaction via upregulation of autophagy in endothelial cells post-AKI by (1) examining if Klotho restores peritubular capillaries with structural analysis (histology and high resolution ?CT), and measurement of renal blood perfusion and oxygenation (functional MRI, fMRI); (2) testing if Klotho blocks endothelial-mesenchymal transition in AKI mice with endothelial tracing markers; (3) confirming if Klotho protects endothelial damage and endothelial-mesenchymal transition in cultured endothelial cells via modulation of autophagy. Aim 1 focuses on basic biology of Klotho effect on autophagy, which builds the basis for other two independent but scientifically interconnected Aims. This renewed proposal was built based on previous 5-year studies and will provide more evidence to support the concept that Klotho upregulates autophagy to inhibit fibrosis, ameliorates rarefaction after AKI and retards AKI-to-CKD progression. Developing new approaches to enhance renal vascular recovery and inhibit fibrosis is the translational objective of the proposal. Bringing Klotho to a therapeutic platform in human AKI is in the proximal future.